CN101280340B - Specific molecular marker capable of identifying sea-tangle female and male gametophytes - Google Patents
Specific molecular marker capable of identifying sea-tangle female and male gametophytes Download PDFInfo
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- CN101280340B CN101280340B CN2008100382212A CN200810038221A CN101280340B CN 101280340 B CN101280340 B CN 101280340B CN 2008100382212 A CN2008100382212 A CN 2008100382212A CN 200810038221 A CN200810038221 A CN 200810038221A CN 101280340 B CN101280340 B CN 101280340B
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Abstract
The invention belongs to the technology field of the gene engineering, in particular relates to a specificity molecule marker which can distinguish laminaria female and male gametophyte. The specificity molecule marker is obtained through the following method: two pairs of primers of SEQ ID No1and SEQ ID No2 are designed according to 5'end flanking sequence of the laminaria female and female gametophyte gene groups, polymerase chain reaction amplification is performed to the deoxyribonucleic acid of the laminaria female and female gametophyte gene groups, and the deoxyribonucleic acid of specificity amplification fragment are reclaimed; large intestine rod competent strain JM109 is cloned through pMD19-T vector to obtain a laminaria female gametophyte specificity molecule marker Rf-763 anda laminaria male gametophyte specificity molecule marker Rm-239, and the sequences thereof are SEQ ID No3 and SEQ ID No4. The genders of the laminaria gametophyte, parthenogenesis laminaria sporophyte parental source and the genders of the offspring thereof can identified through utilizing the specificity molecule marker, and the laminaria gender differentiation in basis of the cell and moleculelevel can be studied.
Description
Technical field
The invention belongs to gene engineering technology field, the specific performance mark that relates to a kind of plant sex signing, relate in particular to and a kind ofly can distinguish that sea-tangle is female, the specific molecular marker of microgametophyte, utilize these molecule markers can fast and effeciently identify the kelp gametophyte sex, also can apply to the evaluation of monogenesis kelp spore body parental source and offspring's sex thereof.
Background technology
For most of plants, sex identification mainly is that carry out aspects such as the formalness, Physiology and biochemistry difference, chemical agent processing, isozyme collection of illustrative plates, differential protein, karyotype, nucleic acid by plant.Along with reaching its maturity of the development of molecular biotechnology, particularly molecular marking technique, make the effect of molecule marker in the plant sex identification seem more and more important.Because molecule marker can directly reflect the difference of plant from the genomic dna level, its mensuration process is not subjected to the influence of development of plants stage and time, greatly increases economic efficiency.
Initiate first-generation dna molecular marker from people such as Grozdicker in 1974---restriction fragment length polymorphism mark (Restriction Fragment Length Polymorphism, RFLP) since, various molecule markers grow up like the mushrooms after rain, up to the present, nearly tens kinds have been developed into.As random amplification product polymorphic dna (RandomlyAmplified Polymorphic DNA, RAPD), simple repeated sequence [(Simple Sequence Repeat, SSR, claim little satellite (Microsatellite) sequence again)], expressed sequence tag (Expressed Sequence Tag, EST), restriction fragment length polymorphism (the Amplified Fragment Length Polymorphism of amplification, AFLP), the grappling simple sequence repeats (Inter-simple Sequence Repeat, ISSR), single nucleotide polymorphism (Single NucleotidePolymorphism, SNP), single strand conformation polymorphism (Single-stranded Conformation Polymorphism, SSCP) etc., these labeling techniques are at the plant molecular genetic map construction, plant genetic diversity analysis and Idioplasm identification, the Main Agronomic Characters assignment of genes gene mapping and map based cloning, aspects such as transgenic plant evaluation and molecular marker assisted selection breeding play a significant role.At present, the molecule marker that is used for the plant identification sex mainly contains RAPD, AFLP, SSR etc.
But on algae, utilize the report of its sex of molecular markers for identification considerably less.In the algae research field, Haring etc. utilize some relevant proterties of chlamydomonas (Chlamydomonas eugametos) sex, make up one and had 15 RAPD marks, the partial linkage collection of illustrative plates of 3 morphology marks and a RFLP mark is for the utilization genetic map research proterties work relevant with property lays the foundation.Martinez etc. utilize RAPD to identify the sex of rhodophyta Gracilaria fragrant plant mentioned in ancient texts (Gracilariagracilis), utilize primer of OPD13 (5 '-TCGTCACCCC-3 ') just simultaneously in female, the male individuality of fragrant plant mentioned in ancient texts, to amplify two difference segments, and successfully be applied to early sex and identify.
Sea-tangle has tangible digenesis life history.The large-scale sporophyte stage is the amphiploid stage, sea-tangle telianthus.The miniature gametophyte stage, promptly from the trip spore diffuse formation female, microgametophyte, and discharge smart, ovum, this is the monoploid stage of sea-tangle, dioecism carries out syngenesis.At present, the report that does not still have relevant sea-tangle male and female temper body specific molecular marker both at home and abroad.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can distinguish that sea-tangle is female, the specific molecular marker of microgametophyte, provide foundation for kelp gametophyte sex identification, monogenesis kelp spore body parental source and offspring's sex identification thereof and from problems such as cell or molecular level research sea-tangle sexual differentiation.
The present invention utilizes specially designed two couples of primer Rf (SEQ.ID.NO1) and primer Rm (SEQ.ID.NO2), and the DNA that extracts in female to sea-tangle respectively, the microgametophyte carries out pcr amplification.The result shows, Rf increases with primer, from sea-tangle megagametophyte genomic dna amplified production, obtain the specific band (seeing shown in Figure 1) of an about 800bp, and, these bands of a spectrum only occur in the amplification of sea-tangle megagametophyte genomic dna, in sea-tangle microgametophyte genomic dna, do not have this amplified band, extraordinary repeatability is arranged.Rm increases with primer, from sea-tangle microgametophyte genomic dna amplified production, obtain the specific band (seeing shown in Figure 2) of an about 250bp, and these bands of a spectrum only occur in the amplification of sea-tangle microgametophyte genomic dna, in the amplification of sea-tangle megagametophyte genomic dna, do not have this amplified band, extraordinary repeatability is arranged.
Through order-checking, the size of sea-tangle megagametophyte specific band is 763bp, and sequence is SEQ.ID.NO3, as sea-tangle megagametophyte specific molecular marker, is designated as Rf-763.The size of sea-tangle microgametophyte specific band is 239bp, and sequence is SEQ.ID.NO4, and the specific molecular marker as the male wine made of broomcorn millet daughter of sea-tangle is designated as Rm-239.
Proposed by the invention can distinguish that sea-tangle is female, the acquisition step of the specific molecular marker of microgametophyte is as follows:
(1) at 17 ± 1 ℃, 30-40 μ mol m
-2s
-1Illumination condition is cultivated female, the male gamete haploid clone of sea-tangle down, collects and extract gametophytic genomic dna;
(2) the sea-tangle 5 '-flanking sequence female, microgametophyte lhcf6 gene that is EU663626 and EU663627 according to the last sequence accession number of GenBank designs two couples of primer SEQ.ID.NO1 and SEQ.ID.NO2, and female to sea-tangle respectively, microgametophyte genomic dna carries out pcr amplification;
(3) reclaim the segmental DNA of specific amplified.Utilization is purchased in the pMD19-T of Takara company carrier, intestinal bacteria (Escherichia coli) competence bacterial strain JM109, clones (connecting conversion reagent box specification sheets according to Takara company); Select white colony, use the PCR method to confirm to insert in the carrier segmental length scale, to contain the segmental bacterium liquid of purpose checks order, obtain two specific molecular markers, be designated as Rf-763 and Rm-239, the former is a sea-tangle megagametophyte specific molecular marker, and the latter is a sea-tangle microgametophyte specific molecular marker, see illustrated in figures 1 and 2ly, their sequence is respectively SEQ.ID.NO3 and SEQ.ID.NO4.
What the present invention proposed distinguishes that sea-tangle is female, the specific molecular marker of microgametophyte, the sex identification that can be used for kelp gametophyte sex identification, monogenesis kelp spore body parental source and offspring thereof also can be used for from problems such as cell or molecular level research sea-tangle sexual differentiation.
Description of drawings
Fig. 1 sea-tangle megagametophyte specific molecular marker Rf-763 electrophoretogram.The M:DL250DNA size criteria; 0 swimming lane: blank; 1-10 swimming lane: megagametophyte; 11-20 swimming lane: microgametophyte.
Fig. 2 sea-tangle microgametophyte specific molecular marker Rm-239 electrophoretogram.The M:DL250DNA size criteria; 0 swimming lane: blank; 1-10 swimming lane: megagametophyte; 11-20 swimming lane: microgametophyte.
Embodiment
1) gets the kelp gametophyte clone that fresh weight is 0.1g.
2) in liquid nitrogen, gently pulverize gametophyte clone last, the 0.6mlCTAB that is transferred to preheating immediately extracts in the damping fluid, contain weightmeasurement ratio in this damping fluid and be 3% CTAB, 1.4M NaCl, 20mMEDTA, 10mM pH and be 7.8 Tris-HCl and weightmeasurement ratio and be 2% mercaptoethanol, mixing 1min on vortex mixing instrument.
3) temperature is bathed 30-60min in 65 ℃ of water-baths, and every 5min action is put upside down light and slowly and shaken 1 time.
4) adding isopyknic volume ratio is 24: 1 chloroform: foreign matter alcohol mixed solution, put upside down mixing 3-5min gently, till emulsion layer occurring.
5) at room temperature 12 000rpm (rev/min) centrifugal 10min, supernatant liquor is transferred in the centrifuge tube of another sterilization.
6) Virahol of adding 2/3 volume-20 ℃ following precooling in centrifuge tube is put upside down mixing, till the appearance precipitation.
7) in-20 ℃ place down about 15min after, the 0-4 ℃ of centrifugal 10min of 8 000rpm down.
8) be deposited in 70% washing with alcohol and in the clean platform of aseptic behaviour after the air seasoning, be dissolved in the TE damping fluid of 200 μ l sterilization, it is 8.0 Tris-HCl that this damping fluid contains 1mM EDTA and 10mM and pH value.
9) treat to dissolve fully the ammonium acetate of the 7.5M that afterwards adds 1/3 volume and the dehydrated alcohol of 2 times of volume-20 ℃ following precoolings.Put upside down mixing, till the appearance precipitation.
10)-20 ℃ place down more than the 30min, 0-4 ℃ down behind the centrifugal 10min of 12 000rpm, precipitate with 70% washing with alcohol again.With in the TE damping fluid of resolution of precipitate in about 40-100 μ l ,-20 ℃ of preservations are standby at last.
11) according to accession number in the GenBank database be that the sea-tangle of EU663626 and EU663627 is female, lhcf6 gene 5 '-two couples of primer SEQ.ID.NO1 of flanking sequence design and SEQ.ID.NO.2 in the microgametophyte, utilize that following reaction system and response procedures are female to sea-tangle, the microgametophyte genomic dna carries out specific amplification.It is 8.3 Tris-HCl damping fluid, 2.5mM Mg that 20 μ l reaction systems comprise 50mM KCl, 10mMpH
2+, 100 μ M dNTPs, 0.2 μ M primer, 1.0U Taq archaeal dna polymerase and 50ng template DNA.Amplification program comprises 94 ℃ of pre-sex change 5min, and then carries out 30 circulations by 94 ℃ of sex change 30s, 57 ℃ of annealing 45s, 72 ℃ of these programs of extension 1min, and last 72 ℃ are extended 10min.Amplification carries out electrophoresis on 1.5% sepharose, observe under the ultraviolet lamp and take pictures.
12) use is purchased in the UNIQ-10 pillar DNA of Shanghai biotechnology company limited glue and is reclaimed test kit, reference reagent box experimental procedure description operation.Kelp gametophyte sex specific amplified product is carried out electrophoresis on 1.5% sepharose, female, male differential fragment and other amplifications are separated as far as possible, cut off differential fragment with clean blade then, put into the 2.0ml centrifuge tube.
13) every 100mg gel adds 700 μ l adsorption-buffering liquid, places 50-60 ℃ of water-bath 15min, and gel is thoroughly melted, and every 2min puts upside down mixing once.
14) sol solution that melts is transferred in the UNIQ-10 post, room temperature is placed 2min, the centrifugal 1min of the speed of 8 000rpm under the room temperature.
15) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, add 500 μ l rinsing liquids, the centrifugal 1min of 8 000rpm under the room temperature.
16) repeating step 15) once.
17) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, the centrifugal 15s of the speed of 12 000rpm under the room temperature.
18) the UNIQ-10 post is put into the centrifuge tube of a new 1.5ml, added 40 μ l lysates in pillar film central authorities, room temperature is placed 2min.
19) the centrifugal 1min of 12 000rpm under the room temperature, the liquid in the centrifuge tube is the dna segment of recovery, be stored in-20 ℃ standby.
20) add 1 μ l pMD19-T plasmid vector in Eppendorf tube, 0.1-0.3pmol is inserted into dna fragmentation, adds deionized water to 5 μ l again.
21) after the connection mixed solution of adding 5 μ l, 16 ℃ of reaction 30min.
22) above-mentioned 10 μ l reaction solutions are added in the escherichia coli jm109 competent cell of 100 μ l, place 30min in the ice.Shake centrifuge tube 2-3 time gently, with abundant mixing.
23) behind the ice bath centrifuge tube changed over to heat shock 90s in 42 ℃ of water-baths, then ice bath 3-5min immediately.
24) the LB liquid nutrient medium of adding 890 μ l, the velocity fluctuation of 37 ℃ of following 150rpm is cultivated 45-60min.Every liter of LB liquid nutrient medium contains the 10g Tryptones, the 5g yeast extract, and 10g NaCl behind the NaOH adjusting pH to 7.0 with 5M, is settled to one liter with single water that steams at last, and sterilization gets final product.
25) get 0.1ml and contain the bacterium liquid that connects product and coat on the LB Agar Plating and cultivate, 37 ℃ of overnight incubation form single bacterium colony.LB agar plate preparation: in every 100ml LB liquid nutrient medium, add 1.5g agar, the 5-bromo-4-chloro-3-indoles-beta galactose glycosides (X-Gal), the isopropylthio-(IPTG) of 2.4mg and the penbritin (Amp) of 10mg that add 4mg when nutrient agar subject to sterilization is cooled to 50 ℃ of left and right sides, be made into X-Gal, IPTG, Amp plate culture medium, shake up the back and fall dull and stereotyped.
26) picking is containing the white colony of growing on the LB solid medium of penbritin after transforming, and is inoculated into the 100 μ g ml that contain of 0.5-1ml
-1In the LB liquid nutrient medium of penbritin.
27) speed of 37 ℃ of following 150rpm concussion overnight incubation.
28) get 100 μ l bacterium liquid in the centrifugal 2min of the speed of 12 000rpm.
29) abandoning supernatant, the Triton X-100 aqueous solution of adding 40 μ l 5% in precipitation.
30) boil 5min, place 5min on ice, the centrifugal 5min of 12 000rpm.
31) get 2 μ l supernatant liquors as template DNA, carry out the PCR reaction.25 μ l PCR primitive reaction systems are formed and are comprised 50mM KCl, and 10mM pH is 8.3 Tris-HCl damping fluid, 2.0mM Mg
2+, 200 μ M dNTPs, M13 forward and oppositely each 0.2 μ M of universal primer, 1.0U Taq archaeal dna polymerase and 30ng template DNA.Basic amplification program comprises 94 ℃ of pre-sex change 3min, and then carries out 30 circulations according to 94 ℃ of sex change 45s, 57 ℃ of annealing 45s, 72 ℃ of programs of extending 1.5min, and last 72 ℃ are extended 10min.Preserve amplified production for 4 ℃.After PCR reaction finishes, get product 6 μ l at 1.5% agarose gel electrophoresis, electrophoretic buffer is 1 * TAE, and this damping fluid comprises 40mM Tris-HAc and 2mM EDTA.Voltage is 5V cm
-1, observe under the ultraviolet lamp and take pictures.
32) will contain the segmental bacterium liquid of purpose delivers to Shanghai bio-engineering corporation and checks order.
Two specific molecular marker Rf-763 and Rm-239, its sequence is respectively SEQ.ID.NO.3 and SEQ.ID.NO.4.SEQ.ID.NO1 (primer Rf):
Forward primer 5 '-GGTCTGTTTGGTCAAGGTTAT-3 '
Reverse primer 5 '-TTTAGGTTGGGTTGGGGATT-3 '
SEQ.ID.NO2 (primer Rm):
Forward primer 5 '-GTGCTTGAGGCTAAAGAAACC-3 '
Reverse primer 5 '-TGGAAGTGGAGATGAGAGAGG-3 '
SEQ.ID.NO3(Rf-763)：
GGTCTGTTTGGTCAAGGTTATTATTGTATTTTCAATAAATTGAAAGTCAGTAAATCC
AATTTTAGTGCATCACCATCCGATAACTTTTGTTTGGAAATCAACTTACCATTTACCT
TGGTTCCATTAGTACTCTTGAGATCTTCAATAAATAAAAGGCGATTTCCATCAAGAA
ATGGGTCTGGCTCAGAAACTACCCGCGCATGTTTTGAGCTCACTGACTGATCTTCTA
TTTGAATATCGCAATCTTTGGCACGGCCTAAAATCGTGACTTCTTCTATAAGCTCCCA
CTTATTAATAATTACATCTTCAACAATTAAAGCTAAATAGGCCATTTTATTCCCACTG
TCTATTGTTTAGTTTTATTCGGATAACAATTTAATGCTAACCAAAGTGATATTATCTT
TGCTACCATTTTTGATTGCCCTTGCAACCATTTTTTGACAAACATCATCGCCGGCTGA
AAGGCTTTGCATAAACTCGGATAATTCTTGCGAGCTAAGACTATCATGTAATCCATC
GGTTGATAATAATAAGCTCGCAGAGATGTGAATCGGTAGTGACTTAACATCAACTTC
AACACCCTTTCTAATGCCGATCGCTTTAGAAAGCATGTTTTTATTATTAGAAGTGTCC
GCTTCCTTTTGAGTTATCGCGCCTTTATCTAATAAGGCTTGAACCATCGTATGGTCAA
CCGTCAATTGAGCCAGATGATTGTTTTTTGCTTCATATAAATAGCATCGAGAATCCC
CAACCCAACCTAAAT
SEQ.ID.NO4(Rm-239)：
GTGCTTGAGGCTAAAGAAACCATGTCCGGATGCTCAATTCATGTAGTCTCAGAAGA
ATATGATAAAGGACCGATTTTAGAACAGGCAACAGTTGACGTACTAGCCTCTGATA
CTCCTGAAGAGCTTGCTGCCAGGGTGTTACAACAAGAACACCTCATCTATCCAAAAG
TAATCGAAACATATTTAAATACACTCAACGAAAACTAATGGCTTTACAACCTCTCTC
ATCTCCACTTCCA
Claims (3)
1. can distinguish that sea-tangle is female, the specific molecular marker of microgametophyte for one kind, the specific molecular marker that it is characterized in that the sea-tangle megagametophyte is Rf-763, its sequence is SEQ.ID.NO3, and the specific molecular marker of sea-tangle microgametophyte is Rm-239, and its sequence is SEQ.ID.NO4.
2. one kind is used to increase that sea-tangle is female, the primer of microgametophyte genomic dna, it is characterized in that being used to increasing the primer of sea-tangle megagametophyte genomic dna to being Rf, sequence is SEQ.ID.NO1, and the primer of the sea-tangle microgametophyte genomic dna that is used to increase is to being Rm, and sequence is SEQ.ID.NO2.
3. as claimed in claim 1ly can distinguish that sea-tangle is female, the extracting method of the specific molecular marker of microgametophyte for one kind, it is characterized in that concrete steps are as follows:
(1) at 17 ± 1 ℃, 30-40 μ mol m
-2s
-1Illumination condition is cultivated female, the male gamete haploid clone of sea-tangle down, collects and extract gametophytic genomic dna;
(2) the sea-tangle 5 '-flanking sequence female, microgametophyte lhcf6 gene that is EU663626 and EU663627 according to the last sequence accession number of GenBank designs two couples of primer SEQ.ID.NO1 and SEQ.ID.NO2, and female to sea-tangle respectively, microgametophyte genomic dna carries out pcr amplification;
(3) reclaim the segmental DNA of specific amplified, utilize pMD19-T carrier, intestinal bacteria competence bacterial strain JM109, clone; Select white colony, use the PCR method to confirm to insert in the carrier segmental length scale, to contain the segmental bacterium liquid of purpose checks order, obtain two specific molecular markers, be designated as Rf-763 and Rm-239, the former is a sea-tangle megagametophyte specific molecular marker, and the latter is a sea-tangle microgametophyte specific molecular marker, and their sequence is respectively SEQ.ID.NO3 and SEQ.ID.NO4.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660647A (en) * | 2012-05-14 | 2012-09-12 | 上海海洋大学 | Specific ISSR-SCAR marker for distinguishing female gameiophytes of larainaria japonica aresch |
| CN102660648B (en) * | 2012-05-14 | 2014-01-22 | 上海海洋大学 | Chromosomal localization for FSML (female-specific marker of Laminaria japonica Aresch)-1488 |
| CN103497951A (en) * | 2013-09-05 | 2014-01-08 | 上海海洋大学 | Kelp gametophyte lhcf 6 gene promoter and application thereof |
| CN106834469B (en) * | 2017-02-15 | 2020-06-09 | 青岛农业大学 | Specific molecular marker of male gametophyte of kelp and application of specific molecular marker |
| CN108034742B (en) * | 2017-12-05 | 2021-07-23 | 山东东方海洋科技股份有限公司 | Method for identifying sex of kelp gametophyte |
| CN112760410B (en) * | 2021-03-11 | 2024-02-02 | 上海海洋大学 | Kelp female gametophyte specific molecular marker FSMSJ-1294 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148115C (en) * | 2000-09-08 | 2004-05-05 | 中国海洋大学 | Gametophyte cloning method of breeding heterotic kelp |
| CN1631118A (en) * | 2004-12-24 | 2005-06-29 | 中国海洋大学 | Kelp breeding method based on gamete selection technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148115C (en) * | 2000-09-08 | 2004-05-05 | 中国海洋大学 | Gametophyte cloning method of breeding heterotic kelp |
| CN1631118A (en) * | 2004-12-24 | 2005-06-29 | 中国海洋大学 | Kelp breeding method based on gamete selection technology |
Non-Patent Citations (1)
| Title |
|---|
| Xiu－Liang Wang et al.DNA fingerprinting of selected Laminaria(Phaeophyta) gametophytes by RAPD markers.Aquaculture.2004,2381-11. * |
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